Our long-term goal is to elucidate the biological functions of the glycosyl moieties on receptors or other glycoconjugates important to the development or functions of the immune system. By combining recent advances in glycoconjugate biochemistry with the genetics, serology, and bio-assays of the murine immune system, we are continuing to take an electric approach, to study the biosynthesis and functions of complex oligosaccharide moieties. Specific Aims for the Next Five-Years Are: 1. To study the biosynthesis, compartmentalization, and functions of the Novel Protein-Saccharide Linkage, O-GlcNAc. We recently discovered a new form of glycosylation that, unlike all other known types, is localized to the cytoplasm and nucleus of the cell. Our plan is to isolate and characterize the glycosyltransferase responsible for O-GlcNAc addition to protein, and to take several different approaches to elucidate the functions of these structures. 2. To study the biosynthesis of the novel C-terminal phosphatidylinositol-Containing Glycolipid Membrane Anchor on Thy- 1 and Other Immunologically Important Receptors. Our plan is to use the tools and knowledge that we have gained from the study of the variant surface glycoprotein (VSG) to elucidate the pathways of biosynthesis of these novel glycolipid anchors on mammalian lymphocyte receptors. 3. To continue to investigate the biochemical and biological basis, and functional significance of glycosylation-site specific oligosaccharide microheterogeneity. Since site-specific oligosaccharide microheterogeneity appears to be highly-controlled, our basis is that it may be a universal way of "fine-tuning" the biological functions of receptors in a way that is directly responsive to the phenotype or metabolic state of the cell. We propose to study site-specific glycosylation in response to development, genetics of the cell, and to determine its origin at the cellular level. 4. To continue to use highly- purified glycosyltransferases as probes of saccharide topography on the surfaces of living cells and in isolated subcellular compartments. As structural probes, these enzymes are analogous to restriction enzymes for molecular biologists, in that there is nearly a unique specific enzyme for every type of glycoside linkage. We plan to extend our studies by continuing to probe the surfaces of developing cells and adult cells in the immune system, as well as the subcellular distribution and re-cycling of glycoconjugates. 5. To continue to collaborate on the gene cloning of glycosyltransferases purified and used in specific aim. 6. Our plan is to provide the purified enzyme, affinity-purified polyclonal antibodies, and the necessary amino acid sequence data for the synthesis of nucleotide probes, as well as for confirmation of clones. These studies are making use of some of the best model systems to address fundamental questions about the roles of glycosylation in developmentally or immunologically important cellular interactions or functions.